Design and expression of IL-15 constructs. (A) Schematic representation of the wild-type and membrane-bound IL-15 constructs (wtIL15 and mbIL15) used in this study. Amp-R, ampicillin resistance gene; IRES, internal ribosome entry site; LTR, long terminal repeat. (B) Expression of IL-15 on the surface of NK cells transduced with mbIL15. Expanded NK cells were transduced with wtIL15, mbIL15, or a vector containing GFP alone (mock). Flow cytometry dot plots illustrate expression of GFP and IL-15, as detected by an anti-IL15 antibody (R&D Systems) and a PE-conjugated goat-anti-mouse secondary antibody (Southern Biotechnology Associates). Percentage of cells (>98% CD56⁺ CD3⁻ NK cells) in each quadrant is shown. (C) Secretion of IL-15 by NK cells transduced with wtIL15. NK cells from 3 different donors were tested in triplicate. Bars indicate mean ± SD of enzyme-linked immunosorbent assay measurements performed in supernatants collected after 24 and 48 hours of culture without IL-2. No IL-15 was detected in the supernatants of mock-transduced NK cells.